Septic Murine Pulmonary Microvascular Endothelial Barrier Dysfunction in vitro is Regulated by TIMP3

Abstract

Sepsis often causes dysfunction of pulmonary microvascular endothelial cells (PMECs) leading to pulmonary edema. Metalloproteinases can regulate endothelial function through processing of cell surface proteins, including adherens junctions proteins, which has been associated with increased permeability. Tissue inhibitor of metalloproteinases 3 (TIMP3) regulates metalloproteinase function in the lung following injury. Thus, we hypothesize that TIMP3 promotes PMEC stability via inhibition of metalloproteinase activity. To test this hypothesis, we used PMECs isolated from WT and Timp3‐/‐mice. We found that TIMP3 levels (mRNA and protein) were decreased in WT PMECs in a time‐dependent fashion under septic conditions (cytomix). Analysis of leak (transendothelial electrical resistance, dextran, and albumin flux) revealed that Timp3‐/‐PMECs had significantly higher permeability under resting conditions (PBS) vs. WT PMECs. Although cytomix treatment significantly increased WT trans‐PMEC permeability, there was no effect on Timp3‐/‐PMEC barrier function. Additionally, the increased basal Timp3‐/‐ PMEC permeability was associated with disrupted surface vascular endothelial‐cadherin localization, which was rescued by treatment with GM6001, a synthetic inhibitor of metalloproteinases. Together, our data suggest that TIMP3 supports normal PMEC barrier function and that septic downregulation of TIMP3 may be an important contributor to septic PMEC barrier dysfunction.